DE102008060332A1 - Microfluidic sorting device with optical tweezers - Google Patents

Abstract

The present invention relates to a sorting apparatus for sorting particles and to a corresponding method. The term particles refers to living cells, cell parts or globules with diameters of a few hundred nanometers to a few micrometers. To provide a sorting device for sorting of particles and an associated sorting method, which overcomes the disadvantages of the known systems and the development of a flexible, fast and kostenitung a miniaturized flow cell with a channel which is laminar flows through a sample solution containing the particles to be sorted in operation , Furthermore, at least one optical detection unit is provided for determining a geometric position of the particles to be sorted. An optical tweeter deflectable by a mirror or an acousto-optic deflector is provided for sequentially sorting the classified particles by selectively moving the particle from the detected position to a predetermined target position in the laminar sample solution stream.

Description

The The present invention relates to a sorting apparatus for Sorting of particles as well as a corresponding procedure. The term particles refers to living cells, cell parts or Beads (so-called "beads") with diameters from a few 100 nanometers to a few micrometers.

The Sorting small particles has been particular in recent years has become very important in biotechnology. It exists an increasing need, functionalized beads, biological Cells or chromosomes by a variety of characteristics to separate from a mixture. For example, cells must often based on their shape and size or based the distribution of a fluorescent dye are separated. These Dyes can be attached to specific molecules which in turn bind to target proteins, or they can characterize a DNA sequence within the nucleus. Other possible Coupling mechanisms and fields of application for fluorescent dyes are of course familiar to the expert.

A interesting property of living cells is their ability to fluorescent proteins (eg green fluorescent proteins, GFP) synthesize, among other things, the successful transfection signaling DNA into a cell. Another example is the Display of the replication potential of hematopoietic Stem cells.

Farther is the analysis of encoded proteins after successful sequencing of the human genome in the context of the human genome project in the foreground Today's research and development. In this context will be Protein microarrays and multiplexed sandwich immunoassays based on binding proteins to beads, the biggest opportunities. Also here fluorescence labeling dominates over other techniques, such as separation by means of magnetic markers.

It is known, small particles with the aid of fluorescence-activated cell sorters, so-called fluorescent activated cell sorters, FACS. In these known FACS devices, the particles in embedded tiny droplets, by means of an electrode charged and then deflected in an electric field after their emission spectrum has been analyzed. In this technique the particles are initially in the fluid stream, then become separated into droplets and then sorted first. This droplet separation is technically relatively consuming.

FACS is well studied, fast and reliable, but this Technology has some serious disadvantages.

To the one the devices require relatively large sample volumes, for another, they can only be through specially trained Staff are serviced and maintained and also provides sterilization a problem. The efficiency of sorting is decreasing Sample volumes of less than 100,000 cells significantly. About that In addition, the acquisition and operating costs of the FACS devices usually too high for smaller laboratories. After all is a portable single-use system based on the FACS technology currently available not foreseeable.

optical Forces for use in sorting small particles in biotechnology a promising alternative With optical traps or optical tweezers, as is common can be called particles in all three spatial directions be moved with high precision. As with this technique no mechanical contact between manipulation device and Particles take place, attachment problems are usually present these proportions of large Meaning are completely avoided. If objective lenses can be used with high numerical aperture high gradient of the electric field and thus high optical Forces are exerted on living tissue, where the overall intensity is kept at a reasonable level can be avoided, so cell damage can be avoided.

These Gentle treatment can be supported by that for the case light with a wavelength in the infrared range not used by the aqueous solution is absorbed inside the cell. Optical traps allow one very fast displacement of particles and allow a parallelization via multiple traps or optical Grid. In addition, the forces can to the trapped particles in a simple manner by the laser power and the wavelength range from Nanonewton to some Femtonewton be set. At the same time, the position with controlled and measured by a nanometer precision.

There are various approaches to combine optical manipulation with microfluidic systems to achieve particle sorting. Miniaturized flow chambers can be produced as individual parts within a few hours, for example by means of so-called soft lithography. In this way, different geometries can be flexibly tested during the development phase. Since the cost of materials and manufacturing tools are relatively low, such chambers can be both cheap and portable Disposable articles, as well as being made as reusable chambers. The small channel dimensions require only small volumes of transport medium and less expensive biochemical reagents.

The Concepts in known microfluidic systems with optical manipulation can be divided into passive and active sorting strategies become. In the case of passive sorting, optical grids or optical line tweezers with asymmetrical intensity profile used. They allow an efficient parallel sorting of Globules, but are based on a distinction based on the Size or refractive index limited.

Nearly all promising active graders work in microfluidic environment, but differ accordingly their particle classification and manipulation strategies. For example is known to particles in a variety of microchannels to sort by temporarily activating a static line trap becomes. In addition, no method uses three-dimensional optical forces what defines optical tweezers.

Furthermore, there are sorting devices in which the particles are hydrodynamically focused within a sorting channel to be sorted by activating a static trap generated by a laser. An example of such an optically switchable optical trap is in Lin et al. Biomed Microdevices (2008) 10: 55-63. "Microfluidic cell sorter / counter utilizing multiple particle tracing technique and optically switching approach." , described.

The Fluorescence of the particles may be similar to a FACS device, be analyzed by that the emission wavelength is detected with a photomultiplier tube. she can but also with a CCD or CMOS camera, measured as a 2D image and analyzed.

When An alternative to optical manipulation could also be one dielectrophoretic sorting can be used. dielectropherotic Forces are based on the same physics as optical forces, but the sorters work with a much larger one Wavelength and field gradients and forces are therefore much lower. As electric fields as well are applied by means of fixed electrodes, is the flexibility low with regard to different sorting schemes.

The Task underlying the present invention consists therefore, a sorter for sorting particles as well as an associated sorting method, which the Disadvantages of the known systems overcomes and development a flexible, fast and cost-effective system allows.

These The object is achieved by the subject matter of the independent patent claims solved. Advantageous developments of the present invention are Subject of the dependent claims.

The The present invention is based on the idea that a sorting device for sorting particles a miniaturized flow cell with a channel that is laminar in operation from one to be sorted Particles containing sample solution flows through is included. Furthermore, an optical detection unit for Determining a geometric position of the particles to be sorted and for optically classifying the particles to be sorted. According to the invention, the sorting device comprises also a dynamic optical tweezer for sequential Sorting the classified particles by selectively moving the Particles from the detected position to a predetermined target position in the laminar sample solution stream. In particular, the Inventive sorting device by the Using a three-dimensional trap capable of the particles to move dynamically in (three-dimensional) space.

With Such an arrangement, particles with the help of a dynamic high-focussed force field are shifted, wherein the entire arrangement works in aqueous solution. By use in a microfluidic environment, the total amount significantly reduced on required reagents.

For the purposes of the present invention, the term "optical tweezers" is understood to mean a highly focused laser which uses the three-dimensional optical gradient force as the physical principle. The radiation pressure is negligible here. This optical gradient force is based essentially on dipole forces and acts most lateral to the beam direction. Ashkin et al. Optics Letters, Vol. 11, no. 5, May 1986, 288-290 , describes in detail the occurring optical forces.

Of the Advantage over using weakly focused Laser radiation consists mainly in that the efficiency at the same Laser power and the effect much higher is. This significantly reduces the risk of cell damage. The use of very high laser powers also has in addition economic disadvantages, such as impairment of laboratory safety or high initial cost of the apparatus.

According to an advantageous embodiment of According to the present invention, the optical detection unit comprises a first light source to illuminate the flow cell and a digital image processing unit to evaluate a transmission image of the flow cell. In this way, a fully automatic operation can be enabled. In response to the detected position of the particles to be sorted, the optical tweezers are respectively targeted to the particle and driven to move the particle.

By the use of a laminar flow causes the displacement of the particle in a defined target position onward transport of the particle in a predetermined output channel. In this way It is possible on a variety of output channels to sort and thus a parallel sorting different To carry out particles from a particle mixture.

For the fully automated further processing of the optical information regarding the particles to be sorted includes the digital Image processing unit according to an advantageous Embodiment of a CCD camera (charged couple device).

The Light source that is used to create a transmitted light image For example, it may include a light emitting diode, LED.

Often If the particles to be detected are whole, often living, cells. To the sorting process for such cells to perform as gently as possible, is in accordance with an advantageous embodiment of present invention adapted the digital image processing unit, the outer shell of the cell and its exact position to detect in the sample solution stream. Based on the noted Position of the cell and in particular its outer shell so can the highly focused laser beam of optical tweezers be directed that he targeted the membrane of the outer shell is directed. In this way, damage to the located inside the cell elements largely avoided.

additionally or alternatively to the analysis based on the visible light can be an evaluation of fluorescence radiation to be sorted by the Particles emitted is done. As already mentioned, This fluorescence radiation can be artificially applied Fluorescence markers are derived or generated by the cell itself be.

Corresponding the fluorescence radiation to be detected must be used as the receiver a detection unit may be provided which is in the sought wavelength range is sensitive.

According to one Advantageous development of the present invention is the sorting device mountable on a standard microscope, so that the Cost of setting up such sorting workstation can be kept as low as possible.

following The present invention is based on preferred embodiments explained in more detail with reference to the accompanying drawings. In this case, the same parts with the same reference numerals and the same Component names provided. Furthermore, features can also or combinations of features from the different ones shown and described Embodiments for themselves inventive or inventive solutions represent.

It demonstrate:

1 a perspective schematic representation of a flow cell during a sorting process for two types of particles;

2 an overview of the sorting device according to the invention when installed in a microscope;

3 a first schematic representation of the forces caused by the optical tweezers on a solid sphere;

4 a second schematic representation of the forces caused by the optical tweezers on a full sphere;

5 a transmitted image on a basophilic leukemia cell of the rat, RBL cell, and representation of the speed components of the optical tweezers in the x and y direction;

6 Sequences of images of a cell at various stages of digital image analysis;

7 a transmitted light representation of a path of a bead with a diameter of 5 microns;

8th a transmitted light representation of a path of an RBL cell manipulated by the optical tweezers.

Regarding 1 Let us first explain the general basic principle of the sorting device according to the invention. A miniaturized flow cell 104 , which consists of a transparent material, has a channel system for flowing through with a sample solution. Here is the main channel 108 from a laminar flow of a sample solution with the injected particles to be sorted 110 flows through. A first input channel A carries the carrier solution while the particles 110 be entered through a second input channel B in the system. The arrow 112 symbolizes the general flow direction. The main channel 108 divides at its end in the flow direction in two output channels, C and D. Of course, the principles of the invention are transferable to more than two output channels, if more components are to be separated accordingly.

As in 1 schematically indicated, according to the invention, the flow cell 104 in the area of the main canal 108 from a dynamic optical tweezers, so a laser beam with strong focus, 114 , penetrated. An infrared laser is focused by a microscope objective with high numerical apparatus (NA). In this way, high field gradients become in the main channel 108 generated. Globules or cell membranes, generally particles 110 , which are in the focus area, experience strong forces transverse to the beam axis, ie in the lateral direction. The arrow 113 which is perpendicular to the flow direction 110 is directed, indicates the deflection direction of the trap transversely to the flow direction. The decisive lateral forces transverse to the beam axis are made possible only by the displacement of the trap.

By the possibility of focusing the optical tweezers 114 made by the lens, opened up for the inventive solution the ability to integrate them in a commercially available transmitted light microscope.

The flow cell 104 is advantageously produced by means of a soft lithography process. An SU-8 negative photoresist is patterned by means of UV light, then polydimethylsiloxane, PDMS, is poured over it and baked. After the structured PDMS has been activated by means of an oxygen plasma, it is connected to a standard slide plate. Connecting hoses are attached to the inlets and outlets of the input and output channels A, B, C, D. In this arrangement, the main channel has a cross-sectional area of, for example, 50 × 50 μm ² , and the flow rate can be adjusted hydrostatically.

Regarding 2 the following is the sorting device according to the invention 100 be explained form according to a first attachable to a commercial microscope execution form. This is partially the lens system of the microscope 102 used.

According to the invention with reference to 1 described flow cell 104 on the stage 106 fixed. In the arrangement drawn here are two light sources 116 and 118 used that alternately the flow cell 104 illuminate. For example, a light source radiating in the green serves as a bright field illumination and is required for the detection of the particle positioning as well as for the control of the optical tweezers.

The second light source 118 can emit radiation in a blue wavelength range to excite fluorescence in the particle. This fluorescence radiation is used for a classification of the particles. In any case, the radiation falling through the tube lens TL by means of a deflection mirror and a filter F2 from a CCD camera 120 recorded and evaluated.

A laser radiating in the infrared wavelength range 122 is part of the optical tweezers. With the help of a scanner arrangement 115 according to the invention comprises a movable mirror SM and a lens SL, the focus of the laser according to the invention within the main channel 108 the flow cell 104 be moved in both the x and in the y direction. The displacement may be, for example, ± 40 μm at a rate of about one kHz. In the 2 not shown is an electronic control unit according to the by the CCD camera 120 obtained information about the position of the particle to be sorted controls the scanner unit to selectively deflect the detected and analyzed particles.

In the 2 The arrangement shown also comprises two beam splitters D1, D2 (so-called dichroic beam splitters), which have a reflective effect for a certain wavelength range but are transparent to other wavelength ranges. Thus, the beam splitter D1 allows the passage of the blue fluorescence excitation radiation of the second light source 118 , but is impermeable to the radiation of the IR laser, so that it is deflected into the interior of the microscope and to the beam splitter D2. The beam splitter D2 reflects both the fluorescence excitation radiation and the light of the laser 122 through the objective lens OL in the main channel of the flow cell 104 , For the bright field radiation of the light source 116 However, the beam splitter D2 is as transparent as for the optionally excited on the particles fluorescence radiation. This radiation is via the microscope mirror M2 to the CCD camera 120 directed.

According to the invention, the focus of the optical tweezers 114 Depending on which geometric position and which type of particle has resulted in the analysis by the CCD camera functioning as an optical detection unit, the particle moves according to the movement and from a detected actual position in the laminar flow of the main channel 108 moved to a desired target position becomes. The movement of the optical tweezers is in principle possible in all three spatial directions x, y and z, wherein a piezoelectric acousto-optic deflector must be used for the deflection of the focus in the z-direction. At the in 1 arrangement shown, for example, lighter first particles shown 109 moved into the output channel C, while the darker second particles 111 be moved in the direction of the output channel D, as by the Auslenkpfeil 113 is indicated.

As already mentioned, but can also do more than just Two varieties of particles are sorted when more than two output channels C, D are provided.

With reference to the 3 and 4 In the following, the forces exerted on the particles to be sorted by the focused radiation of the optical tweezers will be considered in greater detail. The force ratios in these figures are shown for globules of homogeneous material. For such particles with a diameter which is much larger than the wavelength of the optical tweezers, the occurring optical forces can be modeled using the beam optics. These particles are also referred to as Mie particles and assume that although the gradient forces still act, they are more easily illustrated by the momentum transfer of the photons to the object. The photons are not absorbed in the particles, but only reflected and broken at the interfaces.

An arithmetical treatment of the forces occurring on a cell membrane, for example, the essay Robert C. Gauthier: "Computation of the optical trapping force using FDTD based technique", 16 May 2005, Vol. 10, OPTICS EXPRESS 3707-3718 , refer to.

The optical power density is calculated to f → opt = ħg → / Δt where g is the net momentum acting on the particle. Integration over the entire particle, F → _opt = ∫ _A f → dA, provides the total optical force. This must be large enough to overcome the oppositely acting hydrodynamic frictional force F → _frict = 6παη · ν, which is determined by the radius a of the particle, the viscosity η of the liquid and the drawing speed v.

In the presentation of 3 the focus of the laser is at its focal point F inside the spherical particle above the center. The beam is refracted at the interfaces and by vectorial addition one obtains a net force in the direction of the focal point, ie in the 3 up.

The 4 shows a lateral displacement, in which again the particle is pulled in the direction of the focal point, which is symbolized by the leftward arrow g. That is, the particles are always moved toward the focal point of the optical tweezers. The total force on the particle F → _opt = ∫f → dA must, as already mentioned, be large enough to overcome the hydrodynamic frictional force.

The Inventive sorting device is basically suitable for all suspension cells, that is for Cells that are not fixed on a substrate or not in larger associations, such as Chains, happen. In addition to those for the following examples Experiments used rat basophilic leukemic cells, RBL cells are e.g. As well as yeast cells such suspension cells. The achievable speeds are several 100 μm / s.

The optical sorting device according to the invention makes it possible to attack cells only on their membrane due to the strong focusing of the laser. For this purpose, with the aid of the optical detection unit, which in the representation of 2 through the light source 116 and the associated CCD camera 120 is formed, the position of the cell membrane in the laminar flow of the main channel 108 certainly.

5 , shows in the overview a transmitted light image on the main channel with an RBL cell in it and schematically the trapping speeds in the x and y direction. The RBL cell has a radius a and moves through the flow in the direction of flow 112 (assumed here as the x direction) with the velocity V _transport . In order to overcome the lowest possible flow resistance when sorting, according to the invention, the optical tweezers also moves at this speed in the x-direction during the entire sorting process.

At time ➀, the optical tweezers are accelerated in the y-direction until they are deflected by the distance b (time ➁). Then, the optical tweezers pulls at constant speeds in the x and y direction until a sufficient deflection of the particle is achieved in the desired target position. At time ➂, the optical tweezers are decoupled from the particle and transported from the laminar sample solution to the corresponding exit channel. In the arrangement shown, the distance b over which an acceleration of the optical tweezers takes place corresponds approximately to the cell radius a. However, it is clear to a person skilled in the art that other values are also set you can.

6 shows the individual evaluation steps I to IV, which are carried out by a digital image processing. Based on the information obtained, the laser is focused on the membrane of the cell and then deflected by means of the scanner mirror SM to pull the cell, acting on its membrane, to a desired target position in the laminar flow.

The Forces acting on a cell membrane are proportional to the intensity gradient of the radiation. The membrane The cell has a higher refractive index than the cell surrounding solution and also a higher refractive index as the cell interior.

7 shows by the superposition of multiple images the path of a homogeneous bead with a diameter of 5 microns, which is drawn from the input channel side A of the main channel to the output channel side D.

In contrast, shows 8th the ratios for an RDL cell, where speeds of 250 microns / s are achieved here, when the laser power in focus is 28 mW. It is known that laser powers up to 100 mW can be tolerated by the cell without significantly damaging it. With this value of 28 mW laser power, a sorting rate of up to 10 cells per second can be achieved if the cells on the input channel B are dense and continuous.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited non-patent literature

• Lin et al. Biomed Microdevices (2008) 10: 55-63 [0013] "Microfluidic cell sorter / counter utilizing multiple particle tracing technique and optically switching approach"
• - Ashkin et al. Optics Letters, Vol. 11, no. 5, May 1986, 288-290 [0020]
• Robert C. Gauthier: "Computation of the optical trapping force using FDTD based technique", 16 May 2005, Vol. 10, OPTICS EXPRESS 3707-3718 [0052]

Claims (16)

Sorting device for sorting particles comprising: a miniaturized flow cell ( 104 ) with a channel that is laminar in operation by a particle to be sorted ( 110 ) is flowed through the sample solution containing; at least one optical detection unit ( 116 . 118 . 120 ) for determining a geometric position of the particles to be sorted and for optically classifying the particles to be sorted; a deflectable optical tweezer for sequentially sorting the classified particles by selectively moving the particle from the detected position to a predetermined target position in the laminar sample solution stream. Sorting apparatus according to claim 1, wherein the optical Tweezers over a mirror or an acousto-optic Deflector is deflectable. Sorting apparatus according to claim 1 or 2, wherein the optical detection unit comprises a first light source ( 116 ) to scan the flow cell, and a digital image processing unit ( 120 ) to evaluate a transmission image of the flow cell. Sorting apparatus according to claim 3, wherein the digital Image processing unit a charge Couped Device, CCD, camera includes. Sorting apparatus according to claim 3 or 4, wherein the first light source ( 116 ) comprises a light-emitting diode, LED. Sorting device according to at least one of the claims 3 to 5, wherein the digital image processing unit is adapted, a position of an outer area of the particles to be sorted to identify. Sorting apparatus according to claim 6, wherein the dynamic optical tweezers is driven to respond in response to the identified Position of the outer area of the particles to be sorted to be focused on the outdoors. Sorting device according to at least one of the preceding Claims, wherein the optical detection unit for detecting fluorescence-labeled particles is set up. Sorting device according to at least one of claims 3 to 8, wherein the optical detection unit comprises a second light source ( 118 ) disposed separately from the first light source. The sorting device according to at least one of the preceding claims, wherein the dynamic optical tweezers comprise a highly focused capture laser ( 122 ) or a laser diode. Sorting device according to at least one of the preceding Claims, wherein the flow cell at least one feed channel (A, B) and at least two output channels (C, D) comprises and The dynamic optical tweezers are operable to sort Particles corresponding to an output signal of the optical detection unit to move to a selected output channel. Sorting device according to at least one of the preceding claims, which is designed such that it can be attached to a commercially available microscope ( 102 ) is mountable. Method for sorting particles with the following steps: Transporting a particle to be sorted containing sample solution in a laminar sample solution stream; Determine a geometric position and optical classification of the sorting particles; sequential sorting of the classified Particles by targeted movement of the particle by means of a dynamic optical tweezers to a predetermined target position in the laminar Sample solution flow. The method of claim 13, wherein the step of Determining a geometric position and the optical classifying the particle to be sorted comprises: Examining a part the sample solution current and evaluate a transmitted light image by means of a digital image processing unit. The method of claim 14, wherein the digital image processing unit a position of an outer area of the particles to be sorted identified. The method of claim 15, wherein the dynamic optical tweezers in response to the identified position of the Outside area of the particles to be sorted on the outside area is focused.